Fluorescent compounds as sensing agents

ABSTRACT

A method may comprise: exposing a substituted chromone dissolved in a solvent to a sample; taking a fluorescence measurement of the sample after exposure to the substituted chromone; and determining a presence or absence of one or more ions in the sample, a concentration of the one or more ions in the sample, or both based on the fluorescence measurement.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Patent ApplicationNumber PCT/US 2017/019153 filed on Feb. 23, 2017, and U.S. ProvisionalApplication Ser. No. 62/299,321 filed on Feb. 24, 2016, entitled“Fluorescent Compounds as Sensing Agents”, which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to a new class of fluorophores that, in someinstances, shifts emission wavelength upon binding of heavy metal ions.The fluorophores are useful for, for example, detecting the presence ofheavy metal ions in water.

BACKGROUND

Over the past decade, there has been a significant interest influorescence chemical sensing. This has been fueled by advances ininstrumentation and light emitting probes such as fluorophores.Fluorophores, also referred to herein as fluorescent molecules, aremolecules that absorb electromagnetic radiation and emit light. Thewavelength of the emitted light is impacted by many factors, includingthe structure of the molecule, the presence of bound ions, and solventproperties. Researchers seek to develop fluorophores that undergomeasurable changes in their fluorescence in response to conformationalchanges or ion binding. These molecules may then be used as probes toimage, track, and sense such changes.

Fluorophores typically have limitations and drawbacks. For instance, tobe useful as indicators or probes, a fluorophore preferably exhibits acombination of, if not all of, a sufficiently high absorptioncoefficient and quantum yield to absorb radiation from a radiationsource and subsequently fluoresce strongly to allow for detection, anemission frequency that can be discriminated from any backgroundautofluorescence, minimal to no photobleaching, and a large Stokes shiftto filter out the excitation light. It is also important thatfluorophores be low cost and easy to handle for widespread use instandardly-equipped chemical, physical, and cell biology labs, or foruse outside of the lab. Because of these requirements, the developmentof new, useful fluorophores is a difficult task, especially becausethere is not a good process or set of criteria available to predict afluorophore's overall properties.

BRIEF SUMMARY

In some instances, a method may comprise: exposing a substitutedchromone according to Compound 1 dissolved in a solvent to a sample,wherein R₁ is methyl acetate or methyl (2,2-dimethyl) acetate, R₂ ishydroxymethylene, (4-methylphenylsulfonamido)methylene, or((4-methoxyphenyl)sulfamido)methylene, R₃ is hydrogen, methoxy, bromo,chloro, fluoro, methyl, ethyl, or isopropyl, R₄ is hydrogen, methoxy,bromo, chloro, fluoro, methyl, ethyl, or isopropyl, and R₅ is hydrogen,methoxy, bromo, chloro, fluoro, methyl, ethyl, or isopropyl; taking afluorescence measurement of the sample after exposure to the substitutedchromone; and determining a presence or absence of one or more ions inthe sample, a concentration of the one or more ions in the sample, orboth based on the fluorescence measurement.

In some instances, a method may comprise: exposing a substitutedchromone according to Compound 1 dissolved in a solvent to a sample,wherein R₁ is methyl acetate or methyl (2,2-dimethyl) acetate, R₂ ishydroxymethylene, (4-methylphenylsulfonamido)methylene,((4-methoxyphenyl)sulfamido)methylene, or(4-methoxyphenylamido)methylene, R₃ is hydrogen, methoxy, bromo, chloro,fluoro, methyl, ethyl, or isopropyl, R₄ is hydrogen, methoxy, bromo,chloro, fluoro, methyl, ethyl, and isopropyl, and R₅ is hydrogen,methoxy, bromo, chloro, fluoro, methyl, ethyl, or isopropyl; taking afluorescence measurement of the sample; determining a presence orabsence of Pb²⁺ ions in the sample, a concentration of the Pb²⁺ ions inthe sample, or both based on the fluorescence measurement; adding achelating agent to the sample or a source of the sample; and extractingthe chelating agent complexed with the Pb²⁺ ions from the sample or thesource of the sample.

In some instances, a composition may comprise: Compound 1, wherein R₁ ismethyl acetate or methyl (2,2-dimethyl) acetate, R₂ is hydroxymethylene,(4-methylphenylsulfonamido)methylene, or((4-methoxyphenyl)sulfamido)methylene, R₃ is hydrogen, methoxy, bromo,chloro, fluoro, methyl, ethyl, or isopropyl, R₄ is hydrogen, methoxy,bromo, chloro, fluoro, methyl, ethyl, or isopropyl, and R₅ is hydrogen,methoxy, bromo, chloro, fluoro, methyl, ethyl, or isopropyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an inverse electron demand hetero-Diels-Alder (HDA)reaction useful for producing at least some of the fluorophoresdescribed herein.

FIG. 2 illustrates an inverse electron demand HDA reaction for producing(Z)-methyl2-(3-(hydroxymethylene)-4-oxochroman-2-yl)-2-methylpropanoate.

FIG. 3 illustrates three hypothetical emission spectra for a substitutedchromone of the present disclosure.

FIG. 4 illustrates the emission spectra of a substituted chromone(Compound 11 of Table 1) with increasing amounts of Fe³⁺ ions.

FIG. 5 illustrates the emission spectra spectrum of a substitutedchromone (Compound 20 of Table 1) with increasing amounts of Fe³⁺ ions.

FIG. 6 illustrates the emission spectra of a substituted chromone(Compound 11 of Table 1) with increasing amounts of Fe²⁺ ions.

FIGS. 7-10 illustrate the emission spectra of a substituted chromone(Compound 20 of Table 1) in the presence of Pb²⁺ with different solvents(FIG. 7 in methanol, FIG. 8 in 1:1 tetrahydrofuran:methanol, FIG. 9 in3:1 tetrahydrofuran:methanol, FIG. 10 in tetrahydrofuran).

FIGS. 11-12 illustrate the emission spectra of a substituted chromone(Compound 25 of Table 1) in the presence of Pb²⁺ with different solvents(FIG. 11 in 3:1 tetrahydrofuran:methanol and FIG. 12 in 2:1tetrahydrofuran:methanol).

DETAILED DESCRIPTION

The present disclosure relates to a new class of fluorophores based on achromone structure, where the synthesis may include an inverse electrondemand hetero-Diels-Alder (HDA) reaction from readily availablematerials. The structure of the fluorophores is designed with theflexibility to have multiple substituents to the base chromonestructure. By altering the chemical composition and location of thesubstituents, the properties of the substituted chromone may be adjustedincluding, but not limited to, a peak absorption wavelength, a peakemission wavelength, an ability to chelate to metal ions, and aselectivity for such chelation.

Compound 1 illustrates a general structure for a substituted chromone ofthe present disclosure. More specifically, Compound 1 has a chromonestructure with substitutions in the 2 position (R₁), the 3 position(R₂), optionally the 6 position (R₃), optionally the 7 position (R₄),and optionally the 8 position (R₅). Accordingly, for Compound 1, R₁ maybe methyl acetate or methyl (2,2-dimethyl) acetate, R₂ may behydroxymethylene, (4-methylphenylsulfonamido)methylene,((4-methoxyphenyl)sulfamido)methylene, or(4-methoxyphenylamido)methylene, R₃ may be hydrogen, methoxy, bromo,chloro, fluoro, methyl, ethyl, or isopropyl, R₄ may be hydrogen,methoxy, bromo, chloro, fluoro, methyl, ethyl, or isopropyl, and R₅ maybe hydrogen, methoxy, bromo, chloro, fluoro, methyl, ethyl, orisopropyl.

More specifically, Compounds 2-4 provide general structures of thesubstituted chromones of Compound 1 with the R₂ group specified:Compound 2: R₂ is hydroxymethylene, and Compound 3: R₂ is(4-methylphenylsulfonamido)methylene.

Specific exemplary substituted chromones suitable for use in themethods, kits, systems, and compositions described herein areillustrated in Table 1. It should be noted that the specific embodimentsof Table 1 are intended to be exemplary and are in no way limiting tothe scope of Compound 1.

TABLE 1 Name Structure Compound 5 (Z)-methyl 2-(3- (hydroxymethylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 6 (Z)-methyl 2-(6-fluoro-3- (hydroxymethylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 7 (Z)-methyl 2-(6-chloro-3- (hydroxymethylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 8 (Z)-methy-2-(6-bromo-3- (hydroxymethylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 9 (Z)-methyl-2-(3- (hydroxymethylene)-6-methyl-4-oxochroman-2-yl)-2- methylpropanoate

Compound 10 (Z)-methyl 2-(6-ethyl-3- (hydroxymethylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 11 (Z)-methyl-2-(3- (hydroxymethylene)-6-methoxy-4-oxochroman-2-yl)- 2-methylpropanoate

Compound 12 (Z)-methyl-2-(6-chloro-3- (hydroxymethylene)-7-methyl-4-oxochroman-2-yl)-2- methylpropanoate

Compound 13 (Z)-methyl 2-(6,8-dichloro-3- (hydroxymethylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 14 (Z)-methyl 2-(6,8-dibromo-3- (hydroxymethylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 15 (Z)-methyl 2-methyl-2-(3-((4- methylphenylsulfonamido)meth-ylene)-4-oxochroman-2- yl)propanoate

Compound 16 (Z)-methyl2-(6-fluoro-3-((4- methylphenylsulfonamido)meth-ylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 17 (Z)-methyl 2-(6-chloro-3-((4- methylphenylsulfonamido)meth-ylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 18 (Z)-methyl 2-(6-bromo-3-((4- methylphenylsulfonamido)meth-ylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 19 (Z)-methyl 2-methyl-2-(6- methyl-3-((4-methylphenylsulfonamido)meth- ylene)-4-oxochroman-2- yl)propanoate

Compound 20 (Z)-methyl 2-(6-ethyl-3-((4- methylphenylsulfonamido)meth-ylene)-4-oxochroman-2-yl)-2- methylpropanoate

Compound 21 (Z)methyl-2-(6-isopropyl-3- (((4-methylphenyl)sulfonamido)meth- ylene)-4-oxochroman-2-yl)-2-methylpropanoate

Compound 22 (Z)-methyl-2-(6-methoxy-3- (((4-methylphenyl)sulfonamido)meth- ylene)-4-oxochroman-2-yl)-2-methylpropanoate

Compound 23 (Z)methyl-2-(3-(((4- methoxyphenyl)sulfonamido)methylene)-4-oxochroman-2- yl)-2-methylpropanoate

Compound 24 (Z)methyl-2-(3-(((4- methoxyphenyl)sulfonamido)methylene)-6-methyl-4- oxochroman-2-yl)-2- methylpropanoate

Compound 25 (Z)methyl-2-(6-methoxy-3- (((4- methoxyphenyl)sulfonamido)methylene)-4-oxochroman-2- yl)-2-methylpropanoate

Compound 26 (Z)-methyl-2-(6-methoxy-3- (((4- methoxyphenyl)sulfonamido)methylene)-4-oxochroman-2- yl)acetate

According to various embodiments, the substituted chromones of thepresent disclosure may be readily synthesized using organic chemistrytechniques. FIGS. 1-2 illustrate various exemplary synthetic pathwaysthat may be used to produce substituted chromones. It should be notedthat the featured synthetic pathway embodiments are intended to beexemplary and are in no way limiting to the scope of the syntheticpathway suitable for producing substituted chromones described herein.

FIG. 1 illustrates a general inverse-demand hetero-Diels-Alder (HDA)reaction useful for adding the R₁ group of Compound 1 to the 2 positionof the chromone structure. Compound 34 is a chromone structure havingsubstitution at the 3 position that is the precursor to the R₂ group ofCompound 1 (e.g., an aldehyde for the R₂ group of hydroxymethylene or asulfonamide for the R₂ group of ((4-methylphenylsulfonamido)methylene)or ((4-methoxyphenyl)sulfamido)methylene) and optionally substitution inthe aromatic ring of the chromone corresponding to R₃, R₄, and/or R₅,depending on the desired substituted chromone product. Compound 34 isreacted with dimethylsilyl ketene acetal (Compound 35) to produce aDiels-Alder intermediate shown as Compound 36, which has a third ringstructure added to the chromone structure. The reaction betweenCompounds 34 and 35 may be performed at room temperature (or an elevatedtemperature, e.g., up to about 50° C.) in a solvent like dichloromethaneand/or tetrahydrofuran for about 10 minutes to about 2 hours. Uponworkup, Compound 37, which correspond to some embodiments of Compound 1,is produced and isolated.

FIG. 2 illustrates a specific inverse-demand hetero-Diels-Alder (HDA)reaction for producing Compound 9, which is (Z)-methyl2-(3-(hydroxymethylene)-4-oxochroman-2-yl)-2-methylpropanoate. Briefly,3-formylchromone (Compound 38) is reacted with Compound 35 indichloromethane for 30 minutes at room temperature and worked up toproduce Compound 9.

The substituted chromones of Compound 1 with the R₂ group ofhydroxymethylene may have a peak absorption wavelength between about 300nm and about 400 nm and a peak emission wavelength between about 420 nmand about 520 nm. The substituted chromones of Compound 1 with the R₂group of ((4-methylphenylsulfonamido)methylene) or((4-methoxyphenyl)sulfamido)methylene) may have a peak absorptionwavelength between about 330 nm and about 430 nm and a peak emissionwavelength between about 450 nm and about 550 nm. The values for thepeak absorption wavelength and the peak emission wavelength for specificcompounds are provided in Example 1.

In some embodiments, the substituted chromones of the present disclosuremay form a complex with ions where complexing with the ions may cause achange in the peak emission wavelength and/or the emission intensity fora specific wavelength of the corresponding fluorescence. One or both ofthese fluorescent properties may be monitored to determine the presenceof ions and/or the concentration of ions in a sample. Determination ofthe concentration of ions in the sample may be achieved by comparingfluorescent properties (e.g., the amount of and/or change to the peakemission shift and/or emission intensity) to a table, graph, color chartreference, or other mathematical representation (e.g., a mathematicalformula) of a known correlation between concentration and thefluorescent properties.

As illustrated in Example 1, the fluorescence of the substitutedchromones is in the visible spectrum. Accordingly, the changes in peakemission wavelength may be detected visually upon excitation with a longwavelength UV-source (e.g., a 365 nm source) or other suitableexcitation source that excites the substituted chromone, which may be ator near the peak absorption wavelength (e.g., at a wavelength betweenabout 300 nm and about 450 nm). The color emitted may be usedqualitatively to determine if a specific ion is present (e.g., Example8). Alternatively or in combination, the color emitted may be comparedto reference samples or a color chart reference for a more quantitativeanalysis of ion concentration. For example, a color chart referencespecific to the substituted chromone color change range may be used in asimilar way a color chart reference is used with pH paper toqualitatively and/or quantitatively analyze a color change to pH paper.

Measuring the emission intensity may be performed in multiple ways asillustrated in FIG. 3 using a fluorimeter such as a photodiode,phototransistor, avalanche photodiode, photoresistor, or aphotomultiplier tube. FIG. 3 illustrates three hypothetical emissionspectra for a substituted chromone of the present disclosure whereSpectra A is the fluorescence spectra with no ions present, Spectra B isthe fluorescence spectra with a first concentration of ions present, andSpectra C is the fluorescence spectra with a second concentration ofions present greater than the first concentration. In some instances,the emission intensity may be measured at the peak emission wavelengthfor each spectrum taken (e.g., at wavelengths corresponding to points D,E, and F of FIG. 3). Alternatively or in combination therewith, theemission intensity may be measured at a specific emission wavelength,which may be the peak emission wavelength substituted chromones withouthaving ions present (e.g., at wavelength G of FIG. 3) or at a specificwavelength where emission intensity varies with varying ionconcentration (e.g., at wavelength H of FIG. 3). In some instances, theavailable hardware may dictate the methods available for implementation.For example, if a detector for a single wavelength or small window ofwavelengths is available, analysis according to wavelength H may berequired. In some instances, more than one of the foregoing methods maybe implemented.

In some instances, the substituted chromones of the present disclosuremay form a complex with metal ions. Exemplary metal ions may include,but are not limited to, Fe²⁺, Fe³⁺, Pb²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Cr³⁺, Mn²⁺,and As³⁺, and the like, and any combination thereof. When bound to ametal ion, the peak emission wavelength of the substituted chromones mayshift, typically red-shift to longer wavelengths. As described above andas illustrated in Examples 2-5, the change in fluorescence properties(e.g., peak emission wavelength and/or emission intensity) may becorrelated to a concentration of the metal ions.

The substituted chromones of Compound 1 may be dissolved or otherwisedispersed in a solvent at any suitable concentration to observefluorescence visually or detect fluorescence with a fluorimeter, which,in some instances, may be a concentration of about 0.1 micromolar (μM)to about 5 μM or higher depending on the substituted chromone. In someembodiments, a kit, a method, or a system may utilize a stock solutionof the substituted chromones described herein at a concentration ofabout 1 μM to about 5 μM, where for detection of ions and analysis offluorescence the stock solution may be diluted with a solvent (which maybe the solvent of the stock solution or another solvent miscibletherewith).

In some instances, the substituted chromones of the present disclosurewith varying R groups may preferentially bind trivalent metal ions overdivalent metal ions. For example, in some instances, the substituents ofthe substituted chromone may be selected so that the resulting compoundpreferentially chelates with Fe³⁺ over Fe²⁺ as illustrated in Example 4.Such selectivity may be useful in methods, kits, and systems fordetecting iron ion concentrations in biological systems. For example,iron is a strong catalyst that plays a central role in functions such asoxygen transport and catalysis. However, when iron is present in highconcentrations, the strong catalytic nature of iron results in theformation of high-energy, reactive radical intermediates, such asperoxyl, alkoxyl, and thiyl-peroxyl. The free radical intermediates maythen cause cellular damage by oxidizing proteins and altering nucleicacids. Antioxidants may be employed to ameliorate the effects of ironoxidation by finding and binding free radical species. However,antioxidants are rapidly metabolized and excreted and thus cannot berelied upon entirely. Therefore, it is desirable to monitor iron levelsif an accurate approximate concentration of iron in the body is desired.In one preferred embodiment, the concentration of iron ions in abiological system may be detected by observing the peak emissionwavelength and/or the intensity of emission.

In some embodiments, the substituted chromones of the present disclosurewith varying R groups may preferentially bind with specific metal ions.For example, the R groups may be chosen to selectively bind Pb²⁺ orother environmentally relevant ions (e.g., heavy metal ions).Accordingly, some embodiments may involve exposing substituted chromonesto a water sample (e.g., drinking water, waste water effluent from achemical, manufacturing, or nuclear plant, effluent from a watertreatment plant, and the like), an extract from a water sample, extractsfrom soil samples, and the like) and determining the presence or absenceof specific metal ions and/or determining a concentration of thespecific metal ions. As illustrated in Example 8, Compound 25 may beparticularly useful in detecting and measuring the concentration ofPb²⁺. As used herein, the term “extract” refers to the solvent anddissolved/dispersed chemicals after treating a sample with the solvent.For example, a soil sample may be washed with methanol and the methanolextract may be analyzed for ions according to one or more methodsdescribed herein.

In addition to the R groups of the substituted chromones being chosenfor a desired selectivity to oxidation state or specific ions, thesolvent for the substituted chromones may also affect changes to thefluorescent properties, as illustrated in Examples 3, 5, and 6.Exemplary solvents may include, but are not limited to, water, methanol,ethanol, acetic acid, formic acid, acetone, dimethylformamide,acetonitrile, dimethyl sulfoxide, tetrahydrofuran, dichloromethane,ethyl acetate, and miscible combinations thereof. For example, whendetecting Pb²⁺, a preferred solvent system may be a mixture oftetrahydrofuran and methanol at a relative ratio of about 2:1 to about3:1 tetrahydrofuran:methanol. In some instances, the pH of the solventmay be adjusted, which may also affect the changes to the fluorescentproperties of the substituted chromones.

The substituted chromones described herein have unique flexibility andtailorability as fluorophores and may be used in a variety of assays andcorresponding kits and methods. For example, a kit may include (1)substituted chromones and dissolved in one or more solvents and (2) aset of instructions for analyzing the presence/absence of one or moreions, analyzing the concentration of one or more ions, or analyzingboth. Included with the set of instructions, or separate therefrom, maybe a table, graph, color chart reference, or other mathematicalrepresentation for correlating a change in one or more fluorescenceproperties to the concentration of one or more ions.

If a method using the substituted chromones described herein detects thepresence of ions in a sample, some embodiments may further involve usinga chelating agent to extract the ions from the sample or the source ofthe sample. For example, dimercaprol or dimercaptosuccinic acid may beused for chelating As³⁺, Hg²⁺, or Pb²⁺. Also, ethylenediaminetetraacetic acid calcium disodium versenate, 1,2-dichloroethane, ortriethylamine may be used for chelating Pb²⁺. Other chelating agents forthe various ions described herein would be readily derived from the art.Further, combinations of chelating agents may be used for extraction ofa single ion or extraction of multiple ions. After chelation, thechelated ions may be removed by traditional methods including solventextraction, centrifugation, and the like.

In some instances, chelation and extraction of ions may be done with thesubstituted chromones described herein, whether ion detection isperformed or not. That is, some embodiments, optionally in conjunctionwith the fluorescence detection/analysis methods described herein, mayinvolve adding one or more substituted chromones described herein to asample (e.g., a water sample) and then using solvent extraction (e.g.,with 1-octanol) to extract the one or more substituted chromones withthe chelated ions to the solvent layer, for example, as illustrated inExample 10.

Embodiments of the present disclosure include, but are not limited to,Embodiment A, Embodiment B, Embodiment C, and Embodiment D.

Embodiment A is a composition comprising: Compound 1, wherein R₁ ismethyl acetate or methyl (2,2-dimethyl) acetate, R₂ is hydroxymethylene,(4-methylphenylsulfonamido)methylene, or((4-methoxyphenyl)sulfamido)methylene, R₃ is hydrogen, methoxy, bromo,chloro, fluoro, methyl, ethyl, or isopropyl, R₄ is hydrogen, methoxy,bromo, chloro, fluoro, methyl, ethyl, or isopropyl, and R₅ is hydrogen,methoxy, bromo, chloro, fluoro, methyl, ethyl, or isopropyl. In someinstances, Embodiment A may further include one or more of the followingoptional elements: Element 1: wherein Compound 1 is one selected fromthe group consisting of Compound 2, Compound 3, and Compound 4; Element2: wherein Compound 1 is one selected from the group consisting ofCompound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10,Compound 11, Compound 12, Compound 13, Compound 14, Compound 15,Compound 16, Compound 17, Compound 18, Compound 19, Compound 20,Compound 21, Compound 22, Compound 23, Compound 24, Compound 25, andCompound 26; Element 3: wherein Compound 1 is a first Compound 1 and thecomposition further comprises a second Compound 1 that is different thanthe first Compound 1; Element 4: Element 3 and wherein the firstCompound 1 and the second Compound 1 are independently selected from thegroup consisting of: Compound 2, Compound 3, and Compound 4; Element 5:Element 3 and wherein the first Compound 1 and the second Compound 1 areindependently selected from the group consisting of: Compound 5,Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16,Compound 17, Compound 18, Compound 19, Compound 20, Compound 21,Compound 22, Compound 23, Compound 24, Compound 25, and Compound 26;Element 6: wherein the composition further comprises a solvent; Element7: wherein the composition further comprises one or more ions; andElement 8: Element 7 and wherein the one or more ions comprise one ormore selected from the group consisting of: Fe²⁺, Fe³⁺, Pb²⁺, Hg²⁺,Zn²⁺, Cd²⁺, Cr³⁺, Mn²⁺, and As³⁺. Exemplary combination of the foregoingelements include, but are not limited to, Element 6 in combination withElement 1, Element 2, Element 3, Element 4, or Element 5; Element 7 andoptionally Element 8 in combination with Element 1, Element 2, Element3, Element 4, or Element 5; and Elements 6 and 7 and optionally Element8 in combination with Element 1, Element 2, Element 3, Element 4, orElement 5.

Embodiment B is a method comprising: exposing a substituted chromoneaccording to Compound 1 (see Embodiment A optionally including one ormore of Elements 1-8) dissolved in a solvent to a sample; taking afluorescence measurement of the sample after exposure to the substitutedchromone; and determining a presence or absence of one or more ions inthe sample, a concentration of the one or more ions in the sample, orboth based on the fluorescence measurement. In some instances,Embodiment B may further include one or more of the following optionalelements: Element 9: wherein the fluorescence measurement is a firstfluorescence measurement; wherein the method further comprises taking asecond fluorescence measurement of the sample occurs before exposing thesubstituted chromone; and wherein determining the presence or absence ofthe one or more ions in the sample, the concentration of the one or moreions in the sample, or both based on a comparison of the first andsecond fluorescence measurements; Element 10: wherein taking thefluorescence measurement of the sample involves exposing the sample to a365 nm wavelength source and visually inspecting fluorescence from thesample; Element 11: wherein determining the presence or absence of theone or more ions in the sample, the concentration of the one or moreions in the sample, or both is based on a comparison of the fluorescencemeasurement and a color chart reference; Element 12: the method furthercomprising adding a chelating agent to the sample or a source of thesample; and extracting the chelating agent complexed with the one ormore ions from the sample or the source of the sample; Element 13:wherein the sample is from drinking water; a waste water effluent from achemical, manufacturing, or nuclear plant; or an effluent from a watertreatment plant; and Element 14: wherein the sample is an extract from asoil sample. Exemplary combination of the foregoing elements include,but are not limited to, Element 13 in combination with one or more ofElements 9-12; Element 14 in combination with one or more of Elements9-12; Element 9 in combination with one or more of Elements 10-12;Element 10 in combination with one or both of Elements 11 and 12; andElement 11 in combination with Element 12.

Embodiment C is a method comprising: exposing a substituted chromoneaccording to Compound 1 (see Embodiment A and all optional Elements)dissolved in a solvent to a sample; taking a fluorescence measurement ofthe sample; determining a presence or absence of Pb²⁺ ions in thesample, a concentration of the Pb²⁺ ions in the sample, or both based onthe fluorescence measurement; adding a chelating agent to the sample ora source of the sample; and extracting the chelating agent complexedwith the Pb²⁺ ions from the sample or the source of the sample. Someinstances, Embodiment B may further include one or more of the followingoptional elements: Element 9; Element 10; Element 11; Element 13;Element 14; Element 15: wherein the chelating agent is one or moreselected from the group consisting of: dimercaproll, dimercaptosuccinicacid, ethylenediamine tetraacetic acid calcium disodium versenate,1,2-dichloroethane, and trimethylamine; and Element 16: wherein thesolvent comprises one or more selected from the group consisting of:methanol and tetrahydrofuran. Exemplary combination of the foregoingelements include, but are not limited to, Element 13 in combination withone or more of Elements 9, 10, 11, 15, or 16; Element 14 in combinationwith one or more of Elements 9, 10, 11, 15, or 16; Element 9 incombination with one or more of Elements 10, 11, 15, or 16; Element 10in combination with one or more of Elements 11, 15, or 16; Element 11 incombination with one or both of Elements 15 and 16; and Elements 15 and16 in combination.

Embodiment D is a kit comprising the composition of Embodiment Aoptionally including one or more of Elements 1-8; and a correlationbetween a presence or absence of one or more ions in the sample, aconcentration of the one or more ions in the sample, or both and afluorescence measurement (e.g., a table, a graph, a color chartreference, or a mathematical representation for the correlation).Optionally, the kit may further include one or more of: a fluorimeter, alight source (e.g., a 365 nm wavelength source), a solvent separate fromthe composition, and dilution equipment (e.g., a graduated cylinder, apipette, or other fluid measurement tool).

The following examples are given to illustrate the present disclosure.It should be understood, however, that the present disclosure is not tobe limited to the specific conditions or details described in theseexamples.

EXAMPLES Example 1

Table 2 provides a peak absorption wavelength (λ_(abs) maximum), peakemission wavelength (λ_(em) maximum), a fluorescent quantum yield(φ_(F)), and a synthesis yield for the various compounds. The synthesisyield is based on the synthetic pathway illustrated in FIG. 1.

TABLE 2 Synthesis λ_(abs) maximum λ_(em) maximum Yield (nm) (nm) Φ_(F)(%) (%) Compound 5 362 446  6* 77 Compound 6 364 486  2* 87 Compound 11383 482 20* 82 Compound 14 371 451  3* 72 Compound 15 358 479 37* 78Compound 16 367 492 33* 90 Compound 17 367 483 39* 80 Compound 18 343483 34* 88 Compound 19 367 492 70* 80 Compound 19 380 468  8** 80Compound 19 364 479  27*** 80 Compound 20 370 490 73* 70 Compound 20 369496  16** 70 Compound 20 364 481  30*** 70 Compound 21 368 490 71* 93Compound 22 390 529 66* 97 Compound 23 358 479 38* 72 Compound 24 367492 72* 76 Compound 25 392 527 69* 77 Compound 26 386 529 67* 72 Thesolvent is *dichloromethane, **acetonitrile, or ***cyclohexane.

Example 2

Two substituted chromones (Compound 11 and Compound 20) were dissolvedin methanol and exposed to varying amounts of FeCl₃ (i.e., Fe³⁺). FIG. 4illustrates the shift in emission spectrum of Compound 11 withincreasing amounts of Fe³⁺ ions. With about 10 equivalents of Fe³⁺ ions,the shift in emission spectrum is saturated, with a total of about a 30nm red-shift in peak emission wavelength. FIG. 5 illustrates the shiftin emission spectrum of Compound 20 with increasing amounts of Fe³⁺ions. With about 10 equivalents of Fe³⁺ ions, the shift in emissionspectrum is saturated, with a total of about a 50 nm red-shift in peakemission wavelength. Interestingly, when this experiment is repeated ina 1:1 methanol:water as the solvent, Compound 11 still exhibits ared-shift in peak emission wavelength while Compound 20 does not.

Further, in both FIGS. 4 and 5 the intensity of the peak emissionwavelength changes with changing metal ion concentration, which may beused in conjunction with or separate from the red-shift in peak emissionwavelength in determining the concentration of metal ions.

Example 3

Compound 11 was dissolved in various solvents and then exposed tovarying amounts of FeCl₃ (i.e., Fe³⁺). In polar aprotic solvents such astetrahydrofuran, acetonitrile, and acetone, the peak emission wavelengthof Compound 11 did not exhibit an appreciable red-shift. In polar proticsolvents like water, methanol, and mixtures thereof, the peak emissionwavelength of Compound 11 exhibited an appreciable red-shift.

Example 4

Compound 11 was dissolved in methanol and then exposed to varyingamounts of FeCl₃ (i.e., Fe³⁺) or (NH₄)₂Fe(SO₄)₂.6H₂O (i.e., Fe²⁺). Asshown in FIGS. 4 and 6, the peak emission wavelength of Compound 11undergoes a significant red-shift in the presence of Fe³⁺ (FIG. 4) butdoes not exhibit appreciable shift emission wavelength in the presenceof Fe²⁺ (FIG. 6). Therefore, Compound 11 may be used to detect Fe³⁺while in the presence of Fe²⁺. However, both FIGS. 4 and 6 exhibit anintensity decrease for the peak emission wavelength with increasingmetal ion concentration, which may be used in conjunction with orseparate from the red-shift in peak emission wavelength in determiningthe concentration of metal ions.

Example 5

Compound 20 was dissolved in methanol and then exposed to varyingamounts of Pb(NO₃)₂ (i.e., Pb²⁺). As shown in FIGS. 7-10, the peakemission wavelength of Compound 20 undergoes a red-shift and intensitydecrease to differing degrees in the presence of Pb²⁺ depending on thesolvent (FIG. 7 in methanol, FIG. 8 in 1:1 tetrahydrofuran:methanol,FIG. 9 in 3:1 tetrahydrofuran:methanol, FIG. 10 in tetrahydrofuran). Inmethanol, the shift in peak emission wavelength maxes out at 10equivalents but no appreciable shift at 1 equivalent. In contrast, amixed tetrahydrofuran/methanol does have an appreciable shift at 1equivalent but a max shift at a much lower concentration (about 5equivalents for 1:1 tetrahydrofuran:methanol and about 3 equivalents for3:1 tetrahydrofuran:methanol). Further, in THF alone, there is noappreciable shift in peak emission wavelength. Therefore, a kit, method,system, or the like may utilize a specific solvent system for detectionof lead or other ions. Alternatively, and especially where the metal ionconcentration is unknown, a kit, method, system, or the like may utilizea series of solvent systems in parallel to determine a concentration ofmetal ions.

Example 6

A 5 μM sample of Compound 25 in 3:1 tetrahydrofuran:methanol or 2:1tetrahydrofuran:methanol was prepared and exposed to varying amounts ofPb(NO₃)₂ (i.e., Pb²⁺) dissolved in deionized water. As shown in FIGS.11-12 using an excitation wavelength of 370 nm, the peak emissionwavelength of Compound 25 undergoes a red-shift and intensity decreaseto differing degrees in the presence of Pb²⁺ depending on the solvent(FIG. 11 in 3:1 tetrahydrofuran:methanol and FIG. 12 in 2:1tetrahydrofuran:methanol). Further, the overall red shift in the peakemission wavelength is very similar, which visually changes thefluorescence from blue to green.

Example 7

The detection limit Pb(NO₃)₂ (i.e., Pb²⁺) for Compound 25 in 3:1tetrahydrofuran:methanol was tested by exposing samples of 5 μM Compound25 to ever decreasing concentrations of Pb(NO₃)₂ dissolved in deionizedwater. A detection limit where the visual change from blue to green wasdetermined to be about 2.5 μM to about 5 μM Pb²⁺, where the use of afluorimeter would allow for detection of even lower concentrations. Thecolor change observed was almost instantaneous, which illustrates thatkits and methods using the substituted chromones described hereinprovide quick results.

Example 8

Compound 25 shows a selectivity for Pb²⁺ ions over other heavy metalions. Compound 25 was dissolved in 3:1 tetrahydrofuran:methanol and thenexposed to varying amounts of Zn/SO₄ (i.e., Zn²⁺), (NH₄)₂Fe(SO₄)₂.6H₂O(i.e., Fe²⁺), Fe₂O₃ (i.e., Fe³⁺), CuSO₄ (i.e., Cu²⁺), or Pb(NO₃)₂ (i.e.,Pb²⁺) each in deionized water. Upon visual inspection when excited at365 nm, the sample exposed to Pb²⁺ turned green while all other samplesremained blue. Similar results were observed when Compound 20 wastested. This example illustrates that the substituted chromones of thepresent disclosure may be chosen to exhibit a fluorescence color changein the presence of specific ions, which may be useful in kits andmethods for detecting specific ions and their concentration in sampleslike drinking water.

Example 9

A 40 mM solution of Pb(NO₃)₂ (i.e., Pb²⁺) was prepared in tap water andthen diluted with 3:1 tetrahydrofuran:methanol to a lead concentrationof 133 μM. The resultant sample was added to solutions of 5 μM sample ofCompound 25 in 3:1 tetrahydrofuran:methanol. A detection limit where thevisual change from blue to green was determined to be about 2.5 μM toabout 5 μM Pb²⁺, where the use of a fluorimeter would allow fordetection of even lower concentrations. This example illustrates thatthe lead detection can be performed using tap water, which includes avariety of other ions.

Example 10

A 3 mM Pb(NO₃)₂ sample was prepared in deionized water and Compound 25was added to give a green fluorescence. The lead was then extracted fromthe water using either 1-octanol or 2-octanol each with 1 equivalent(relative to Compound 25) of triethylamine, which turned the octanolportion green. After washing thrice with 1-octanol, 99% of the Pb²⁺ hadbeen extracted from the water. After washing twice with 2-octanol, 83%of the Pb²⁺ had been extracted from the water.

Example 11

A lead-free soil sample was collected, placed into a Buchner funnel, andsoaked with 1 mL of 0.15 M Pb(NO₃)₂ in water to introduce lead to thesoil. As a control sample, the same was performed with the lead-freesoil without exposure to Pb²⁺. After soaking, the soil samples werewashed with 50 mL of methanol. A sample from each filtrate was dilutedin 3:1 tetrahydrofuran:methanol and exposed to Compound 25. The extractfrom the soil sample exposed to Pb²⁺ exhibited a color change from blueto green. At the same dilution amount, the soil sample not exposed toPb²⁺ exhibited no color change. This example illustrates that thesubstituted chromones described herein may be useful in detecting ionsfrom soil extracts.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities, percentages orproportions, and other numerical values used in the specification andclaims, are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that can vary depending upon thedesired properties sought to be obtained by the present invention. It isnoted that, as used in this specification and the appended claims, thesingular forms “a,” “an,” and “the,” include plural references unlessexpressly and unequivocally limited to one reference. As used herein,the term “include” and its grammatical variants are intended to benon-limiting, such that recitation of items in a list is not to theexclusion of other like items that can be substituted or added to thelisted items.

Any patent, patent application, publication, or other disclosurematerial, in whole or in part, that is said to be incorporated byreference herein is incorporated herein only to the extent that theincorporated material does not conflict with existing definitions,statements, or other disclosure material set forth in this disclosure.As such, and to the extent necessary, the disclosure as explicitly setforth herein supersedes any conflicting material incorporated herein byreference. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material set forth hereinwill only be incorporated to the extent that no conflict arises betweenthat incorporated material and the existing disclosure material.

What is claimed is:
 1. A method comprising: exposing a substitutedchromone according to Compound 1 dissolved in a solvent to a sample,wherein R₁ is methyl acetate or methyl (2,2-dimethyl) acetate, R₂ ishydroxymethylene, (4-methylphenylsulfonamido)methylene, or((4-methoxyphenyl)sulfamido)methylene, R₃ is hydrogen, methoxy, bromo,chloro, fluoro, methyl, ethyl, or isopropyl, R₄ is hydrogen, methoxy,bromo, chloro, fluoro, methyl, ethyl, or isopropyl, and R₅ is hydrogen,methoxy, bromo, chloro, fluoro, methyl, ethyl, or isopropyl,

taking a fluorescence measurement of the sample after exposure to thesubstituted chromone; and determining a presence or absence of one ormore ions in the sample, a concentration of the one or more ions in thesample, or both based on the fluorescence measurement.
 2. The method ofclaim 1, wherein the fluorescence measurement is a first fluorescencemeasurement; wherein the method further comprises taking a secondfluorescence measurement of the sample occurs before exposing thesubstituted chromone; and wherein determining the presence or absence ofthe one or more ions in the sample, the concentration of the one or moreions in the sample, or both based on a comparison of the first andsecond fluorescence measurements.
 3. The method of claim 1, whereintaking the fluorescence measurement of the sample involves exposing thesample to a 365 nm wavelength source and visually inspectingfluorescence from the sample.
 4. The method of claim 1, whereindetermining the presence or absence of the one or more ions in thesample, the concentration of the one or more ions in the sample, or bothis based on a comparison of the fluorescence measurement and a colorchart reference.
 5. The method of claim 1 further comprising: adding achelating agent to the sample or a source of the sample; and extractingthe chelating agent complexed with the one or more ions from the sampleor the source of the sample.
 6. The method of 1, wherein the one or moreions comprise one or more selected from the group consisting of: Fe²⁺,Fe³⁺, Pb²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Cr³⁺, Mn²⁺, and As³⁺.
 7. The method ofclaim 1, wherein the sample is from drinking water; a waste watereffluent from a chemical, manufacturing, or nuclear plant; or aneffluent from a water treatment plant.
 8. The method of claim 1, whereinthe sample is an extract from a soil sample.
 9. A method comprising:exposing a substituted chromone according to Compound 1 dissolved in asolvent to a sample, wherein R₁ is methyl acetate or methyl(2,2-dimethyl) acetate, R₂ is hydroxymethylene,(4-methylphenylsulfonamido)methylene,((4-methoxyphenyl)sulfamido)methylene, or(4-methoxyphenylamido)methylene, R₃ is hydrogen, methoxy, bromo, chloro,fluoro, methyl, ethyl, or isopropyl, R₄ is hydrogen, methoxy, bromo,chloro, fluoro, methyl, ethyl, and isopropyl, and R₅ is hydrogen,methoxy, bromo, chloro, fluoro, methyl, ethyl, or isopropyl,

taking a fluorescence measurement of the sample; determining a presenceor absence of Pb²⁺ ions in the sample, a concentration of the Pb²⁺ ionsin the sample, or both based on the fluorescence measurement; adding achelating agent to the sample or a source of the sample; and extractingthe chelating agent complexed with the Pb²⁺ ions from the sample or thesource of the sample.
 10. The method of claim 9, wherein Compound 1 is


11. The method of claim 9, wherein Compound 1 is


12. The method of claim 9, wherein the chelating agent is one or moreselected from the group consisting of: dimercaproll, dimercaptosuccinicacid, ethylenediamine tetraacetic acid calcium disodium versenate,1,2-dichloroethane, and trimethylamine.
 13. The method of claim 9,wherein the solvent comprises one or more selected from the groupconsisting of: methanol and tetrahydrofuran.
 14. The method of claim 9,wherein the fluorescence measurement is a first fluorescencemeasurement; wherein the method further comprises taking a secondfluorescence measurement of the sample occurs before exposing thesubstituted chromone; and wherein determining the presence or absence ofthe one or more ions in the sample, the concentration of the one or moreions in the sample, or both based on a comparison of the first andsecond fluorescence measurements.
 15. The method of claim 9, whereintaking the fluorescence measurement of the sample involves exposing thesample to a 365 nm wavelength source and visually inspectingfluorescence from the sample.
 16. The method of claim 9, wherein thesample is from drinking water, a waste water effluent from a chemical,manufacturing, or nuclear plant, or an effluent from a water treatmentplant.
 17. The method of claim 9, wherein the sample is an extract froma soil sample.
 18. A composition comprising: Compound 1, wherein R₁ ismethyl acetate or methyl (2,2-dimethyl) acetate, R₂ is hydroxymethylene,(4-methylphenylsulfonamido)methylene, or((4-methoxyphenyl)sulfamido)methylene, R₃ is hydrogen, methoxy, bromo,chloro, fluoro, methyl, ethyl, or isopropyl, R₄ is hydrogen, methoxy,bromo, chloro, fluoro, methyl, ethyl, or isopropyl, and R₅ is hydrogen,methoxy, bromo, chloro, fluoro, methyl, ethyl, or isopropyl


19. The composition of claim 18, wherein Compound 1 is


20. The method of claim 18, wherein Compound 1 is